This invention relates to the production of proteins of interest by eucaryotic cells transformed with selectable markers. In particular, it is concerned with obtaining high yields of secreted, commercially useful proteins such as tissue plasminogen activator from cultured eucaryotic cells which have been cotransformed with genes coding for the protein and for a selectable marker.
The following definitions are supplied in order to facilitate the understanding of this case. To the extent that the definitions vary from meanings circulating within the art, the definitions below are to control.
Amplification means the process by which cells produce gene repeats within their chromosomal DNA.
Cotransformation means the process of transforming a cell with more than one exogenous gene foreign to the cell, one of which confers a selectable phenotype on the cell.
Downstream means the direction going towards the 3' end of a nucleotide sequence.
An enhancer is a nucleotide sequence that can potentiate the transcription of genes independent of the identity of the gene, the position of the sequence in relation to the gene, or the orientation of the sequence.
A gene is a deoxyribonucleotide sequence coding for a given mature protein. For the purposes herein, a gene shall not include untranslated flanking regions such as RNA transcription initiation signals, polyadenylation addition sites, promoters or enhancers.
A selection gene is a gene that confers a phenotype on cells which express the gene as a detectable protein.
A selection agent is a condition or substance that enables one to detect the expression of a selection gene.
Phenotype means the observable properties of a cell as expressed by the cellular genotype.
A product gene is a gene that encodes a protein product having desirable characteristics such as diagnostic or therapeutic utility.
Genotype means the genetic information contained within a cell as opposed to its expression, which is observed as the phenotype.
Ligation is the process for forming a phosphodiester bond between the 5' and 3' ends of two DNA strands. This may be accomplished by several well known enzymatic techniques, including blunt end ligation by T4 DNA ligase.
Orientation refers to the order of nucleotides in a DNA sequence. An inverted orientation of a DNA sequence is one in which the 5' to 3' order of the sequence in relation to another sequence is reversed when compared to a point of reference in the DNA from which the sequence was obtained. Such points of reference can include the direction of transcription of other specified DNA sequences in the source DNA or the origin of replication of replicable vectors containing the sequence.
Transcription means the synthesis of RNA from a DNA template.
Transformation means changing a cell's genotype by the cellular uptake of exogenous DNA. Transformation may be detected in some cases by an alteration in cell phenotype. Transformed cells are called transformants. Pre-transformation cells are referred to as parental cells.
Translation means the synthesis of a polypeptide from messenger RNA.
The transformation of higher, i.e. non-fungal, eucaryotic cells with genes capable of conferring selectable phenotypes has received increasing interest as a method for detecting transformed cells containing amplified genes responsible for selectable phenotypes.
Eucaryotic transformation is in general a well-known process, and may be accomplished by a variety of standard methods. These include the use of protoplast fusion, DNA microinjection, chromosome transfection, lytic and nonlytic viral vectors (For example, Mulligan et al., "Nature"(London) 277:108-114 [1979], cell-cell fusion (Fournier et al., "Proc. Nat. Acad. Sci."74:319-323 [1977], lipid structures (U.S. Pat. No. 4,394,448) and cellular endocytosis of DNA precipitates (Bachetti et al., "Proc. Nat. Acad. Sci. 74:1590-1594 [1977].
Transformation which is mediated by lytic viral vectors is efficient but is disadvantageous for a number of reasons: The maximum size of transfected DNA is limited by the geometry of viral capsid packing, the exogenous genes which are frequently deleted during viral replication, and the requirement for helper virus or specialized hosts, also host cells must be permissive, and the hosts are killed in the course of viral infection.
Some nonlytic viral transformations are based on the transcription and translation of virus vectors which have been incorporated into a cell line as a stable episome. These systems generally require unique cell lines and suffer from a number of disadvantages. See "Trends in Biochemical Sciences", June 1983, pp. 209-212.
On the other hand, other transformations in which extrachromosomal DNA is taken up into the chromosomes of host cells have been characterized by low frequencies of transformation and poor expression levels. These initial difficulties were ameliorated by transformation with genes which inheritably confer selectable phenotypes on the small subpopulation of cells that are in fact transformed. The entire population of transformed cells can be grown under conditions favoring cells having acquired the phenotype, thus making it possible to locate transformed cells conveniently. Thereafter, transformants can be screened for the capability to more intensely express the phenotype. This is accomplished by changing a selection agent in such a way as to detect higher expression.
Selection genes fall into three categories: detectably amplified selection genes, dominant selection genes, and detectably amplified dominant selection genes.
Detectably amplified selection genes are those in which amplification can be detected by exposing host cells to changes in the selection agent. Detectably amplified genes which are not dominant acting generally require a parental cell line which is genotypically deficient in the selection gene. Examples include the genes for asparagine synthetase; aspartate transcarbamylase; (Kemp et al., "Cell"9:541 [1976], adenylate deaminase (DeBatisse et al., "Mol and Cell Biol."2(11):1346-1353 [1982] mouse dihydrofolate reductase (DHFR) and, with a defective promoter, mouse thymidine kinase (TK).
Dominant selection genes are those which are expressed in transformants regardless of the genotype of the parental cell. Most dominant selection genes are not detectably amplified because the phenotype is so highly effective in dealing with the selection agent that it is difficult to discriminate among cell lines that have or have not amplified the gene. Examples of dominant selection genes of this type include the genes for procaryotic enzymes such as xanthine-guanine phosphoriboxyltransferase (Mulligan et al., "Proc. Nat. Acad. Sci."78[4]:2072-2076 [1981] and aminoglycoside 3--phosphotransferase (Colbere-Garapin et al., "J. Mol. Biol.", 150:1-14 [1981].
Some dominant selection genes also are detectably amplified. Suitable examples include the mutant DHFR gene described by Haber et al., "Somatic Cell Genet."4:499-508 [1982], cell surface markers such as HLA antigens and genes coding for enzymes such as specific esterases that produce fluorescent or colored products from fluorogenic or chromogenic substrates as is known in the art.
Detectably-amplified, dominant selection genes are preferred for use herein. It should be understood that a dominant selection gene in some cases can be converted to a detectably amplified gene by suitable mutations in the gene.
Selection genes at first were of limited commercial utility. While they enabled one to select transformants having the propensity to amplify uptaken DNA, most selection genes produced products of no commercial value. On the other hand, genes for products which were commercially valuable generally did not confer readily selectable (or even detectable) phenotypes on their transformants. This would be the case, for example, with enzymes or hormones which do not provide transformed cells with unique nutrient metabolic or detoxification capabilities. Most proteins of commercial interest fall into this group, e.g. hormones, proteins participating in blood coagulation and fibrinolytic enzymes.
Subsequently it was found that eucaryotic cells having the propensity to be transformed with and amplify the selection gene would do the same in the case of the product gene. By following the selection gene one could identify a subpopulation of transformant cells which coexpress and coamplify the product gene along with the selection gene. It has been the practice to culture the transformants in the presence of the selection agent and to conclude that transformants having increased expression of the selection gene will also show increased expression of the product gene. Axel et al., U.S. Pat. No. 4,399,216 use the term cotransformation to describe the process of transforming a cell with more than one different gene, whether by vector systems containing covalently linked or unlinked genes, and in the latter case whether the genes are introduced into host cells sequentially or simultaneously. Cotransformation should "allow the introduction and stable integration of virtually any defined gene into cultured cells"(Wigler et al., "Cell", 16:777-785, [1979], and "by use of the cotransformation process it is possible to produce eucaryotic cells which synthesize desired proteinaceous and other materials"(U.S. Pat. No. 4,399,216, column 3, lines 37-42.)